Vibrio cholerae causes a severe, dehydrating diarrhea in humans. Strains of V. cholerae 01 are divided into two biotypes, classical and E1 Tor. Major virulence factors for this organism include cholera toxin and the toxin-coregulated pilus (TCP). Expression of these virulence factors is regulated in classical V. cholerae by environmental signals such as temperature and pH, and dependent on a cascade of regulatory proteins in which ToxR and ToxS activate transcription of toxT, and ToxT activates expression of cholera toxin, TCP and other virulence genes. In classical V. cholerae, transcription of toxT also requires TcpP and TcpH, which act synergistically with ToxR and ToxS. Frameshift mutations in tcpH have been suggested as a possible mechanism of phase variation in the coordinate expression of cholera virulence genes. Transcription of tcpPH in classical V. cholerae is regulated by temperature and pH independently of ToxR or ToxT, suggesting that TcpP and TcpH may be the couple between environmental signals and expression of virulence factors. V. cholerae is an excellent model system for studying the interaction of a bacterial pathogen with the human host, and particularly regulation of virulence gene expression in response to in vivo signals. The LONG-TERM GOAL of these studies is to use this well-established model system, and a combination of genetic, biochemical, and in vivo experiments, to advance our understanding of how virulence genes are regulated in response to the environment in the human host. There are four SPECIFIC AIMS in the present proposal: (1) analyze the differences in activation of the toxT promoter by TcpP and TcpH between the two biotypes of V. cholerae; (2) study the molecular mechanisms of transcriptional regulation of tcpPH in response to environmental signals; (3) examine the expression of tcpPH in vivo, and test the hypotheses that frameshift mutations in tcpH from "on" to "off" arise late during in vivo infection and that frameshift mutations from "off" to "on" are specifically selected for in vivo at the onset of infection; (4) identify selected other genes in V. cholerae which are necessary for transcription of toxT, focussing on new genes involved in this regulation in conjunction with TcpPH and/or that relate to the specific differences in environmental regulation between the two biotypes.